PI(3)P formation on the SCV is sensitive to pharmacologic inhibitors of PI3-kinase, but PI(3,4)P₂ and PI(3,4,5)P₃ production at invasion ruffles is not. (A) Cells were transfected with PH(Akt)-GFP and infected with WT S. typhimurium in the presence of LY294002. (B, C, and E) Phosphoinositide levels were quantified by HPLC in control cells and after infection with WT in the absence or presence of LY294002, as indicated. The amount of PI(3,4)P₂ (B), PI(3,4,5)P₃ (C), and PI(3)P (E) is given as the percentage of PI in the same sample. (D) Cells were transfected with 2FYVE-GFP and infected with WT in the presence of LY294002. The inset is enlarged from the indicated area (dashed box). Data in B, C, and E are means ± SEM of five separate experiments. Levels of significance are indicated by p- values. Cells in A and D were analyzed by confocal microscopy and correspond to <15 min of infection. Bars, 10 μm.